DESC:FIELD OF INVENTION
The current invention relates to the field of plant breeding. The current invention more specifically relates to methods and compositions for identifying and selecting maize plants with enhanced resistance to Northern leaf blight and/or Exserohilum, by using molecular markers.
BACKGROUND
Maize (Zea mays L.) is one of the most important cereal crops worldwide with a rapidly growing demand in developed as well as developing countries.
Food and agriculture Organization of the United Nations (FAO) predicts that the world’s population is expected to grow to almost 10 billion by 2050, boosting agricultural demand – in a scenario of modest economic growth – by some 50 percent compared to 2013.A meta-analysis of 1 090 studies on yields (primarily wheat, maize, rice and soybeans) under different climate change conditions indicates that climate change may significantly reduce yields in the long run (FAO. 2017. The future of food and agriculture – Trends and challenges. Rome). Moreover, many bacterial, viral and fungal pathogens also pose risk to production levels of major cereals such as maize.
There are a large number of fungal pathogens which cause leaf diseases in maize (Zea mays L.). The fungus which can cause by far the most damage under tropical and also under temperate climatic conditions, such as those in large parts of Europe and North America as well as in Africa and India, is known as Helminthosporium turcicum or synonymously as Exserohilum turcicum. H. turcicum is the cause of the leaf spot disease known as “Northern Corn Leaf Blight” (NCLB) or “Northern Leaf Blight” (NLB), which can occur in epidemic proportions during wet years, attacking vulnerable maize varieties and causing a great deal of damage and loss of yield of 30% or more over wide areas. Northern corn leaf blight is prevalent throughout the world and is known to cause even more than 50% yield losses in many instances. In comparison with America and Europe the productivity of maize in India is low due to a number of biotic and abiotic stresses. The crop is affected by a number of fungal, bacterial and viral diseases. Among the fungal diseases turcicum leaf blight/ northern leaf blight caused by Exserohilum turcicum is one the important foliar disease-causing severe reduction in grain and fodder yield to the tune of 16 - 98% (Ref 8: Singh, R., & Ram, L.).
Symptoms of NCLB can range from cigar-shaped lesions on the lower leaves to complete destruction of the foliage, thereby reducing the amount of leaf surface area available for photosynthesis. A reduction in photosynthetic capability leads to a lack of carbohydrates needed for grain fill, which impacts grain yield. Mid-altitude regions of the tropics, about 900-1600 m above sea level, have a particularly favourable climate for northern leaf blight, as dew periods are long and temperatures moderate. However, northern leaf blight can also lead to heavy yield losses in temperate environments too, such as in the United States, during wet seasons, particularly if the infection is established on the upper leaves of the plant by the silking stage. NCLB reduces the grain yield of maize considerably all over the world.
The fungus Exserohilum turcicum (Et) survives over less favourable temperature and humidity conditions as mycelia and conidia on maize residues left on the soil surface. The conidia are transformed into resting spores, and during warm, moist weather, new conidia are produced and then carried by wind or rain to lower leaves of young maize plants. Infection requires the presence of water on the leaf surface for 6-18 hours and a temperature of between 18° and 30°C. If infection occurs, lesions develop within 7-12 days and produce new conidia, which spread the infection to secondary sites.
Disease management strategies for NCLB include crop rotation, destruction of old maize residues by tillage, and fungicide application, all of which are aimed at reducing the fungal burden.
Among several management options available, cultivation of resistant cultivars is the most practical and cost-effective approach in the management of diseases. Genetics of resistance to northern corn leaf blight suggest that resistance is complex and polygenic in nature with both major and minor QTL (quantitative trait loci). Traditionally, selection for resistance to foliar diseases in maize is practiced through conventional breeding, where resistant genotypes are selected under high disease pressure locations, and susceptible genotypes can be eliminated before harvest. However, such conventional breeding is time-consuming and not so feasible due to the complex nature of resistance to northern leaf blight in maize, and dependency on natural favourable conditions for disease development. Therefore, development of molecular tools to assist conventional breeding efforts to breed resistant cultivars is very important for disease management of northern leaf blight. Identification of quantitative trait loci (QTL) is one such tool to help in marker-assisted selection (MAS) of resistant genotypes.
Molecular breeding is nowadays the method of choice for the utilization of molecular (DNA-based) tools, including markers, to enhance the efficiency of the plant breeding process (Ref 5: Li, Zhu et al; Ref 4: Lema and Melese).
Identification of novel molecular markers associated with any desirable trait is a complex process. Identifying molecular markers linked with NCLB resistance in maize can pave the way for convenient and less time-consuming molecular breeding of NCLB resistant maize plants.
The current invention discloses novel SNP markers linked with NCLB resistance in maize plants, and a method of identifying, and selecting maize plants with NCLB resistance.
SUMMARY
The current invention discloses novel SNP markers linked with NCLB resistance in maize plants, and a method of identifying, and selecting maize plants with NCLB resistance.
One embodiment of the current invention is a SNP marker for identifying a maize plant or maize germplasm exhibiting resistance to northern leaf blight or Exserohilum, wherein the SNP marker comprises presence of a favourable SNP marker allele at a SNP marker locus and wherein the favourable SNP marker allele is selected from the group consisting of : SNP1 which comprises a C to A nucleotide substitution at position no 101 in SEQ ID NO: 1(RLMZNB8), SNP2 which comprises an A to G substitution at position 101 in SEQ ID NO: 3 (RLMZNB9); SNP3 which comprises a G to T substitution at position 101 in SEQ ID NO: 5 (RLMZNB10); SNP4 which comprises T to A substitution at position 101 in SEQ ID NO: 7 (RLMZNB11) , SNP5 which comprises T to C substitution at position 101 in SEQ ID NO: 9 (RLMZNB1) ; SNP6 which comprises C to T substitution at position 101 in SEQ ID NO: 11 (RLMZNB2) and SNP7 which comprises C to A substitution at position 101 in SEQ ID NO: 13 (RLMZNB12).
In one embodiment, the current invention encompasses a method of identifying a maize plant or germplasm that exhibits resistance to northern leaf blight or Exserohilum, the method comprising the steps of:
detecting in a maize plant the presence of at least one favourable SNP marker allele as disclosed herein, wherein the favourable SNP marker allele is associated with resistance to northern leaf blight. In one embodiment, the method further comprises the steps of : (a) obtaining DNA from the maize plant or germplasm; and (b) analysing the DNA from step (a) for presence of any of the SNP markers as disclosed herein.
In one embodiment, the invention encompasses a method of identifying a maize plant or germplasm that exhibits resistance to northern leaf blight or Exserohilum, wherein the method comprises the step of detecting in a maize plant the presence of a favourable SNP marker allele selected from the group consisting of SNP1, SNP2, SNP3 and SNP4 as disclosed herein, wherein the favourable SNP marker allele is associated with resistance to northern leaf blight.
One embodiment of the current invention is a method of selecting a maize plant or germplasm that exhibits resistance to northern leaf blight or Exserohilum, the method comprising the step of selecting the plant identified by the method as disclosed herein.
One embodiment of the current invention is a method of identifying a maize plant that exhibits resistance to northern leaf blight or Exserohilum, the method comprising the steps of: (a) detecting in a maize plant at least one QTL or marker locus associated with resistance to northern leaf blight and/or Exserohilum, wherein the at least one QTL comprises: (i) a first QTL (QTL1) comprising at least one SNP marker allele at a locus within a maize chromosomal interval comprising and flanked by SEQ IN NO: 15 and SEQ ID NO: 16 on chromosome 8, (ii) a second QTL (QTL2) comprising at least one marker allele at a locus within a maize chromosomal interval comprising and flanked by SEQ ID NO: 17 and SEQ ID NO: 18 on chromosome 5; or (iii) a third marker locus comprising a marker allele at a locus within a maize chromosomal interval comprising and flanked by SEQ ID NO: 19 and SEQ ID NO: 20 on chromosome 3.
One embodiment of the current invention is a method for identifying a maize plant or germplasm comprising the first QTL, the second QTL or third QTL linked to resistance to northern leaf blight and/or Exserohilum as disclosed herein, wherein said method comprises the following steps: (a) providing a sample of genomic DNA from a maize plant or germplasm; and (b) detecting in the sample of genomic DNA the presence of at least one SNP marker linked to: (i) the first QTL wherein said SNP marker is selected from the group consisting of SNP1, SNP2, SNP3, and SNP4; (ii) the second QTL wherein said SNP marker is selected from the group consisting of SNP5 and SNP6; or (iii) the third marker locus wherein said SNP marker is SNP7.
The current invention encompasses a method of identifying a maize plant that exhibits resistance to northern leaf blight or Exserohilum, the method comprising the steps of: (a) detecting in a maize plant the presence of at least one resistant SNP marker allele on a SNP marker locus wherein the at least one SNP marker locus is located within a maize chromosomal interval comprising and flanked by (i) SEQ IN NO: 15 and SEQ ID NO: 16 on chromosome 8; (ii) SEQ ID NO: 17 and SEQ ID NO: 18 on chromosome 5; or (iii) SEQ ID NO: 19 and SEQ ID NO: 20 on chromosome 3. In one embodiment, the method further comprises the steps of: (a) isolating DNA from the maize plant or germplasm; and (b) analyzing the isolated DNA for presence of at least one resistant SNP marker allele on a SNP marker locus wherein the at least one SNP marker locus is located within a maize chromosomal interval comprising and flanked by (i) SEQ IN NO: 15 and SEQ ID NO: 16 on chromosome 8; (ii) SEQ ID NO: 17 and SEQ ID NO: 18 on chromosome 5; or (iii) SEQ ID NO: 19 and SEQ ID NO: 20 on chromosome 3
In one embodiment, the method disclosed herein comprises the steps of: detecting in a maize plant the presence of at least one resistant SNP marker allele on a SNP marker locus wherein the at least one SNP marker locus is located within a maize chromosomal interval comprising and flanked by SEQ IN NO: 15 and SEQ ID NO: 16 on chromosome 8.
BRIEF DESCRIPTION OF FIGURES AND DRAWINGS
Fig. 1 shows GWAS QTLs Manhattan plot for NCLB tolerance.
Table 1 gives the description of sequences disclosed in the current application.
DETAILED DESCRIPTION
The current invention discloses novel SNP markers for identifying and selecting maize plants exhibiting resistance to Northern corn leaf blight.
Definitions:
As used herein, the terms “Maize” and “corn” are used interchangeably herein, and refer to a plant of the Zea mays L. ssp. Mays.
As used herein, the term “maize plant” includes whole maize plants, maize plant cells, maize plant protoplast, maize plant cell or maize tissue culture from which maize plants can be regenerated, maize plant calli, maize plant clumps and maize plant cells that are intact in maize plants or parts of maize plants, such as maize seeds, maize cobs, maize flowers, maize cotyledons, maize leaves, maize stems, maize buds, maize roots, maize root tips and the like.
“Enhanced resistance” refers to increased level of resistance against a particular pathogen, more than one pathogen.
“Exserohilum turcicum” is the fungal pathogen that induces northern leaf blight infection. The fungal pathogen is also referred to herein as Exserohilum or Et.
As used herein, a “polymorphism” is a variation in the DNA between two or more individual plants within a population. A polymorphism preferably has a frequency of at least 1 % in a population. A useful polymorphism can include a single nucleotide polymorphism (SNP), a simple sequence repeat (SSR), or an insertion/deletion polymorphism, also referred to herein as an “indel”.
As used herein, the term “allele” refers to one of two or more different nucleotide sequences that occur at a specific locus.
An allele is “associated with” a trait when presence of the particular allele is part of or linked to a DNA sequence or allele is correlated with the expression of the trait.
An allele “negatively” correlates with a trait when it is linked to it and when presence of the allele is an indicator that a desired trait or trait form will not occur in a plant comprising the allele.
An allele “positively” correlates with a trait when it is linked to it and when presence of the allele is an indicator that the desired trait or trait form will occur in a plant comprising the allele.
As used herein, a “favourable allele” or “desired allele” is the allele at a particular locus that confers, or contributes to, an agronomically desirable phenotype, examples of such traits may be disease resistance, resistance to herbicides, etc; the favourable allele allows the identification of plants with that agronomically desirable phenotype. A favourable allele of a marker is a marker allele that segregates with the favourable phenotype.
As used herein, the “favourable allele” is also used interchangeably with “tolerant allele” , since presence of the favorable allele is associated with resistance or tolerance to northern corn leaf blight.
As used herein, an “unfavourable allele” of a marker is a marker allele that segregates with the unfavourable plant trait. Presence of the unfavourable allele can be used for identifying and /or removing plants during line improvement or line development program. The unfavourable allele may or may not be the reference allele.
As used herein, the term “locus” refers to a position on a chromosome, for example the position/ location on a chromosome where a nucleotide, gene, sequence, or marker is located.
As used herein, the term “marker locus” refers to a specific chromosome location in the genome of a species where a specific marker can be found.
Broadly, closely linked loci such as a marker locus and a second locus can display an inter-locus recombination frequency of 10% or less, preferably about 9% or less, still more preferably about 8% or less, yet more preferably about 7% or less, still more preferably about 6% or less, yet more preferably about 5% or less, still more preferably about 4% or less, yet more preferably about 3% or less, and still more preferably about 2% or less.
High-resolution mapping of QTLs may be used to develop reliable markers for marker-
assisted selection (at least <5 cM but ideally <1 cM away from the gene/ linked trait/ second locus) and also to discriminate between a single gene or several linked genes (Ref 2: Collard, B.C.Y., et al).
Two loci that are localized to the same chromosome, and at such a distance that recombination between the two loci occurs at a frequency of less than 10% (e.g., about 9 %, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %, 0.75%, 0.5%, 0.25%, or less) are also said to be “proximal to” or “linked” each other. In some cases, two different markers can have the same genetic map coordinates. In that case, the two markers are in such close proximity to each other that recombination occurs between them with such low frequency that it is undetectable.
As used herein, the term “molecular markers” or “Genetic markers” refers to nucleic acids that are polymorphic in a population. The term includes nucleic acid sequences complementary to the genomic sequences, such as nucleic acids used as probes. Markers corresponding to genetic polymorphisms between members of a population can be detected by methods well- established in the art. These include, e.g., PCR-based sequence specific amplification methods, detection of restriction fragment length polymorphisms (RFLP), detection of isozyme markers, detection of polynucleotide polymorphisms by allele specific hybridization (ASH), detection of amplified variable sequences of the plant genome, detection of self-sustained sequence replication, detection of simple sequence repeats (SSRs), detection of single nucleotide polymorphisms (SNPs), or detection of amplified fragment length polymorphisms (AFLPs). Well established methods are also known for the detection of expressed sequence tags (ESTs) and SSR markers derived from EST sequences and randomly amplified polymorphic DNA (RAPD).
As used herein, the term, “marker allele”, used interchangeably with the term “allele of a marker locus”, can refer to one of a plurality of polymorphic nucleotide sequences found at a marker locus in a population.
As used herein, the term “Marker assisted selection” (of MAS) refers to a process by which individual plants are selected based on marker genotypes. The particular marker genotypes may be linked to specific desirable agronomic traits.
As used herein, the term “Marker assisted counter-selection” refers to a process by which marker genotypes are used to identify plants that will not be selected, allowing them to be removed from a breeding program or planting.
As used herein, the terms “foreground selection” and “forward breeding selection” are used interchangeably, and refer to selecting plants having the marker/ favourable allele of the donor parent at the target locus.
As used herein, the term, the term “haplotype” is the genotype of an individual at a plurality of genetic loci, i.e. a combination of alleles. A haplotype is defined as the combination of alleles for different polymorphisms that occur on the same chromosome, hence the genetic loci described by a haplotype are physically and genetically linked. The term “haplotype” can refer to alleles at a particular locus, or to alleles at multiple loci along a chromosomal segment.
As used herein, the term “marker haplotype” refers to a combination of marker alleles at a marker locus.
As used herein, the term “complement” refers to a nucleotide sequence that is complementary to a given nucleotide sequence.
As used herein, the term “contiguous DNA” refers to an uninterrupted stretch of genomic DNA represented by partially overlapping pieces or contigs.
As used herein, the term “heterogeneity” is used to indicate that individuals within the group differ in genotype at one or more specific loci.
As used herein, a centimorgan (“cM”) is a unit of measure of recombination frequency. One cM is equal to a 1% chance that a marker at one genetic locus will be separated from a marker at a second locus due to crossing over in a single generation.
As used herein, the term “chromosomal interval” designates a contiguous linear span of genomic DNA on a single chromosome. The genetic elements or genes located on a single chromosomal interval are physically linked. The size of a chromosomal interval is not particularly defined or limited. In some aspects, the genetic elements located within a single chromosomal interval are genetically linked, typically with a genetic recombination distance of, for example, less than or equal to 20 cM, or alternatively, less than or equal to 10 cM. Thus, two genetic elements within a single chromosomal interval undergo recombination at a frequency of less than or equal to 20% or 10%.
As used herein, the term “closely linked”, means that recombination between two linked loci occurs with a frequency of equal to or less than about 10% (i.e., are separated on a genetic map by not more than 10 cM). Thus, the closely linked loci co-segregate at least 90% of the time.
SNPs disclosed herein can be detected by any of the methods known in art, examples of which include, but are not limited to, any DNA sequencing, and PCR-based sequence specific amplification methods. Some DNA sequencing methods, have the advantage of being able to detect a series of linked SNP alleles that constitute a haplotype.
As used herein, the term “probe” refers to a nucleic acid sequence or molecule that can be used to identify the presence of a specific DNA or protein sequence; e.g., a nucleic acid probe that is complementary to a marker locus sequence, through nucleic acid hybridization.
As used herein, the term “fragment” refers to a portion of a nucleotide sequence.
As used herein, the term “phenotype”, “phenotypic trait”, or “trait” refer to the observable expression of a gene or series of genes. The phenotype can be observable to the naked eye, or by any other means of evaluation known in the art, e.g., weighing, counting, measuring (length, width, angles, etc.), microscopy, biochemical analysis, or an electromechanical assay.
In some cases, a phenotype is directly controlled by a single gene or genetic locus, i.e., a “single gene trait” or a “simply inherited trait”. In the absence of large levels of environmental variation, single gene traits can segregate in a population to give a “qualitative” or “discrete” distribution, which means that the phenotype falls into discrete classes. In other cases, a phenotype is the result of several genes and can be considered a “multigenic trait” or a “complex trait”.
As used herein, the term “crossed” or “cross” refers to a sexual cross and involves the fusion of two haploid gametes via pollination to produce diploid progeny (e.g., cells, seeds or plants). The term encompasses both the pollination of one plant by another and selfing (or self-pollination, e.g., when the pollen and ovule are from the same plant).
As used herein, the term “backcrossing” refers to the process whereby hybrid progeny are repeatedly crossed back to one of the parents. In a backcrossing scheme, the “donor” parent refers to the parental plant with the desired gene/genes, locus/loci, or specific phenotype to be introgressed. The “recipient” parent (used one or more times) or “recurrent” parent (used two or more times) refers to the parental plant into which the gene or locus is being introgressed.
As used herein, the term “elite line” refers to any line that has resulted from breeding and selection for superior agronomic performance.
As used herein, the term “genetic map” refers to a representation of genetic linkage relationships among loci on one or more chromosomes (or linkage groups) within a given species, generally depicted in a diagrammatic or tabular form. For each genetic map, distances between loci are measured by how frequently their alleles appear together in a population (their recombination frequencies). Alleles can be detected using DNA or protein markers, or observable phenotypes. A genetic map is a product of the mapping population, types of markers used, and the polymorphic potential of each marker between different populations. Genetic distances between loci can differ from one genetic map to another. Information can be correlated from one genetic map to another using common markers. One of ordinary skill in the art can use common marker positions to identify positions of markers and other loci of interest on each individual genetic map. The order of loci does change between maps, although frequently there may be small changes in marker orders due to reasons such as markers detecting alternate duplicate loci in different populations, differences in statistical approaches used to order the markers, novel mutation or laboratory error.
A “physical map” of the genome is a map showing the liner order of identifiable landmarks, including genes, markers etc; on chromosomal DNA. The distances between landmarks on a physical map are absolute, and are measured in base pairs, or isolated and overlapping contiguous genetic fragments. A physical map, in contrast to a genetic map, is not based on frequency of genetic recombination.
As used herein, the term “genetic map location” is a location on a genetic map relative to surrounding genetic markers on the same linkage group where a specified marker can be found within a given species.
As used herein, the term “Germplasm” refers to genetic material of or from an individual (e.g., a plant), a group of individuals (e.g., a plant line, variety or family), or a clone derived from a line, variety, species, or culture, or more generally, all individuals within a species or for several species (e.g., maize germplasm collection or Andean germplasm collection). In general, germplasm provides genetic material with a specific molecular makeup that provides a physical foundation for some or all of the hereditary qualities of an organism or cell culture.
As used herein, the term “haploid” refers to a plant that has a single set (genome) of chromosomes.
As used herein, the term “hybrid” refers to the progeny obtained between the crossing of at least two genetically dissimilar parents.
As used herein, the term “inbred” refers to a line that has been bred for genetic homogeneity.
As used herein, the term “indel” refers to an insertion or deletion, wherein one line may be referred to as having an inserted nucleotide or piece of DNA relative to a second line or the second line may be referred to as having a deleted nucleotide or piece of DNA relative to the first line.
As used herein, the term “introgression” refers to the transmission of a desired allele of a genetic locus from one genetic background to another. For example, introgression of a desired allele at a specified locus can be transmitted to at least one progeny via a sexual cross between two parents of the same species, where at least one of the parents has the desired allele in its genome.
As used herein, the term “linkage” is used to describe the degree with which one marker locus is associated with another marker locus or some other locus. The linkage relationship between a molecular marker and a locus affecting a phenotype is given as a “probability” or “adjusted probability”.
As used herein, the term “linkage disequilibrium” (or LD) refers to a non-random segregation of genetic loci or traits (or both). In either case, linkage disequilibrium implies that the relevant loci are within sufficient physical proximity along a length of a chromosome so that they segregate together with greater than random (i.e., non- random) frequency. Markers that show linkage disequilibrium are considered linked. Linked loci co-segregate more than 50% of the time, e.g., from about 51 % to about 100% of the time. In other words, two markers that co-segregate have a recombination frequency of less than 50% (and by definition, are separated by less than 50 cM on the same linkage group.) As used herein, linkage can be between two markers, or alternatively between a marker and a locus affecting a phenotype.
A marker locus can be "associated with" (linked to) a trait. The degree of linkage of a marker locus and a locus affecting a phenotypic trait is measured, e.g., as a statistical probability of co-segregation of that molecular marker with the phenotype (e.g., an F statistic or LOD score).
As used herein, “linkage equilibrium” describes a situation where two markers independently segregate, i.e., sort among progeny randomly. Markers that show linkage equilibrium are considered unlinked (whether or not they lie on the same chromosome).
The “logarithm of odds (LOD) value” or “LOD score” (Ref 7: Risch, (1992)) is used in genetic interval mapping to describe the degree of linkage between two marker loci. A LOD score of three between two markers indicates that linkage is 1000 times more likely than no linkage, while a LOD score of two indicates that linkage is 100 times more likely than no linkage. LOD scores greater than or equal to two may be used to detect linkage. LOD scores can also be used to show the strength of association between marker loci and quantitative traits in “quantitative trait loci” mapping. In this case, the LOD score’s size is dependent on the closeness of the marker locus to the locus affecting the quantitative trait, as well as the size of the quantitative trait effect.
As used herein, the term, “probability value” or “p-value” is the statistical likelihood that the particular combination of a phenotype and the presence or absence of a particular marker allele is random. Thus, the lower the probability score, the greater the likelihood that a locus and a phenotype are associated. The probability score can be affected by the proximity of the first locus (usually a marker locus) and the locus affecting the phenotype, plus the magnitude of the phenotypic effect (the change in phenotype caused by an allele substitution). In some aspects, the probability score is considered “significant” or “nonsignificant”. In some embodiments, a probability score of 0.05 (p=0.05, or a 5% probability) of random assortment is considered a significant indication of association. However, an acceptable probability can be any probability of less than 50% (p=0.5). For example, a significant probability can be less than 0.25, less than 0.20, less than 0.15, less than 0.1, less than 0.05, less than 0.01, or less than 0.001.
As used herein, the term, “production marker” or “production SNP marker” refers to a marker that has been developed for high-throughput purposes. Production SNP markers are developed to detect specific polymorphisms and are designed for use with a variety of chemistries and platforms.
As used herein, the term, “quantitative trait locus” or “QTL” refers to a region of DNA that is associated with the differential expression of a quantitative phenotypic trait in at least one genetic background, e.g., in at least one breeding population. The region of the QTL encompasses or is closely linked to the gene or genes that affect the trait in question.
Three widely-used methods for detecting QTLs are single-marker analysis, simple interval mapping and composite interval mapping (Ref 2: Collard, B.C.Y et al). Single-marker analysis (also ‘single-point analysis’) is the simplest method for detecting QTLs associated with single markers. The statistical methods used for single-marker analysis include t-tests, analysis of variance (ANOVA) and linear regression. Linear regression is most commonly used because the coefficient of determination (R2) from the marker explains the phenotypic variation arising from the QTL linked to the marker. An “allele of a QTL” (or “QTL allele”) can comprise multiple genes or other genetic factors within a contiguous genomic region or linkage group. An allele of a QTL can be defined by a haplotype within a specified window wherein said window is a contiguous genomic region that can be defined, and tracked, with a set of one or more polymorphic markers. The haplotype is “then “defined by” the unique fingerprint of alleles at each marker within the specified window.
An individual QTL may “also be described as ‘major’ or ‘minor’. This definition is based on the proportion of the phenotypic variation explained by a QTL (based on the R2 value): major QTLs will account for a relatively large amount (e.g. >10%) and minor QTLs will usually account for <10%. Sometimes, major QTLs may refer to QTLs that are stable across environments whereas minor QTLs may refer to QTLs that may be environmentally sensitive, especially for QTLs that are associated with disease resistance.
As used herein, the term “reference sequence” or a “consensus sequence” refers to a defined sequence used as a basis for sequence comparison. The reference sequence for the SNP markers disclosed herein refer to sequences obtained by / from Maize B73-Reference-GRAMENE_v3 (Ref 9: Tello-Ruiz, M. K. et al).
As used herein, the terms “agronomic traits”, and “plant trait or characteristic” are used interchangeably and refer to the traits and associated genotype that ultimately lead to higher yield but encompass any plant characteristic that can lead to higher plant health and yield, such as herbicide resistance, emergence vigour, vegetative vigour, stress tolerance, disease resistance or tolerance, herbicide resistance, branching, flowering, seed set, seed size, seed density, standability, threshability and the like.
The current invention encompasses resistance to northern corn blight as the desirable trait for the maize plants being selected by the method disclosed herein.
Marker-assisted selection (MAS):
Molecular markers can be used in a variety of plant breeding applications A molecular marker that demonstrates linkage with a locus affecting a desired phenotypic trait provides a useful tool for the selection of the trait in a plant population. This is very useful where the phenotype is hard to assay, for example, disease resistance traits. Since DNA marker assays are less laborious and less time and space- consuming than field phenotyping, much larger populations can be assayed, increasing the chances of finding a recombinant with the target segment from the donor line moved to the recipient line.
The closer the linkage, the more useful the marker, as recombination is less likely to occur between the marker and the gene causing the trait, which can result in false positives. Having flanking markers decreases the chances that false positive selection will occur as a double recombination event would be needed.
The ideal situation is to have a marker in the gene itself, so that recombination cannot occur between the marker and the gene. Such a marker is called a’ perfect marker’.
Embodiments:
The present invention relates to maize plants having increased pathogen resistance or tolerance, in particular increased resistance or tolerance to pathogens causing Northern Corn Leaf Blight, that is, Exserohilum turcicum (also known as Helminthosporium turcicum). The current invention encompasses molecular markers for identifying and selecting maize plants with enhanced resistance to northern corn blight. The invention further relates to methods for generating such maize plants, as well as the maize plants generated by the methods disclosed herein. The invention further relates to the use of such maize plants in controlling pathogen infestation. The invention also relates to maize plants or plant parts thereof, obtained by or obtainable by the method as described herein, as well as maize plants or plant parts thereof comprising the polynucleotide sequences described herein.
Table 1: Description of sequences
SNP marker Allele SEQ ID NO
SNP1 (chromosome 8, QTL1) Unfavourable 1
Favourable (resistant/ tolerant) 2
SNP2 (chromosome 8, QTL1) Unfavourable 3
Favourable (resistant/ tolerant) 4
SNP3 (chromosome 8, QTL1)
Unfavourable 5
Favourable (resistant/ tolerant) 6
SNP4 (chromosome 8, QTL1)

Unfavourable 7
Favourable (resistant/ tolerant) 8
SNP5 (chromosome 5 , QTL2)

Unfavourable 9
Favourable (resistant/ tolerant) 10
SNP6 (chromosome 5 , QTL2)
Unfavourable 11
Favourable (resistant/ tolerant) 12
SNP7 (chromosome 3 , QTL3)
Unfavourable 13
Favourable (resistant/ tolerant) 14
Left flanking sequence on chromosome 8 15
Right flanking sequence on chromosome 8 16
Left flanking sequence on chromosome 5 17
Right flanking sequence on chromosome 5 18
Left flanking sequence on chromosome 3 19
Right flanking sequence on chromosome 3 20

The current invention discloses novel SNP markers linked with NCLB resistance in maize plants, and a method of identifying, and selecting maize plants with NCLB resistance.
One embodiment of the current invention is a SNP marker for identifying a maize plant or maize germplasm exhibiting resistance to northern leaf blight or Exserohilum, wherein the SNP marker comprises presence of a favourable SNP marker allele at a SNP marker locus and wherein the favourable SNP marker allele is selected from the group consisting of : SNP1 which comprises a C to A nucleotide substitution at position no 101 in SEQ ID NO: 1(RLMZNB8), SNP2 which comprises an A to G substitution at position 101 in SEQ ID NO: 3 (RLMZNB9); SNP3 which comprises a G to T substitution at position 101 in SEQ ID NO: 5 (RLMZNB10); SNP4 which comprises T to A substitution at position 101 in SEQ ID NO: 7 (RLMZNB11) , SNP5 which comprises T to C substitution at position 101 in SEQ ID NO: 9 (RLMZNB1) ; SNP6 which comprises C to T substitution at position 101 in SEQ ID NO: 11 (RLMZNB2) and SNP7 which comprises C to A substitution at position 101 in SEQ ID NO: 13 (RLMZNB12).
In one embodiment, the current invention encompasses a method of identifying a maize plant or germplasm that exhibits resistance to northern leaf blight or Exserohilum, the method comprising the steps of: a) detecting in a maize plant the presence of at least one favourable SNP marker allele as disclosed herein, wherein the favourable SNP marker allele is associated with resistance to northern leaf blight. In one embodiment, the method further comprises the steps of : (a) obtaining DNA from the maize plant or germplasm; and (b) analysing the DNA from step (a) for presence of any of the SNP markers as disclosed herein.
In one embodiment, the invention encompasses a method of identifying a maize plant or germplasm that exhibits resistance to northern leaf blight or Exserohilum, wherein the method comprises the step of detecting in a maize plant the presence of a favourable SNP marker allele selected from the group consisting of SNP1, SNP2, SNP3 and SNP4 as disclosed herein, wherein the favourable SNP marker allele is associated with resistance to northern leaf blight.
One embodiment of the current invention is a method of selecting a maize plant or germplasm that exhibits resistance to northern leaf blight or Exserohilum, the method comprising the step of selecting the plant identified by the method as disclosed herein.
One embodiment of the current invention is a method of identifying a maize plant that exhibits resistance to northern leaf blight or Exserohilum, the method comprising the steps of: (a) detecting in a maize plant at least one QTL or marker locus associated with resistance to northern leaf blight and/or Exserohilum, wherein the at least one QTL comprises: (i) a first QTL (QTL1) comprising at least one SNP marker allele at a locus within a maize chromosomal interval comprising and flanked by SEQ IN NO: 15 and SEQ ID NO: 16 on chromosome 8, (ii) a second QTL (QTL2) comprising at least one marker allele at a locus within a maize chromosomal interval comprising and flanked by SEQ ID NO: 17 and SEQ ID NO: 18 on chromosome 5; or (iii) a third marker locus comprising a marker allele at a locus within a maize chromosomal interval comprising and flanked by SEQ ID NO: 19 and SEQ ID NO: 20 on chromosome 3.
One embodiment of the current invention is a method for identifying a maize plant or germplasm comprising the first QTL, the second QTL or third QTL linked to resistance to northern leaf blight and/or Exserohilum as disclosed herein, wherein said method comprises the following steps: (a) providing a sample of genomic DNA from a maize plant or germplasm; and (b) detecting in the sample of genomic DNA the presence of at least one SNP marker linked to: (i) the first QTL wherein said SNP marker is selected from the group consisting of SNP1, SNP2, SNP3, and SNP4; (ii) the second QTL wherein said SNP marker is selected from the group consisting of SNP5 and SNP6; or (iii) the third marker locus wherein said SNP marker is SNP7.
The current invention encompasses a method of identifying a maize plant that exhibits resistance to northern leaf blight or Exserohilum, the method comprising the steps of: (a) detecting in a maize plant the presence of at least one resistant SNP marker allele on a SNP marker locus wherein the at least one SNP marker locus is located within a maize chromosomal interval comprising and flanked by (i) SEQ IN NO: 15 and SEQ ID NO: 16 on chromosome 8; (ii) SEQ ID NO: 17 and SEQ ID NO: 18 on chromosome 5; or (iii) SEQ ID NO: 19 and SEQ ID NO: 20 on chromosome 3. In one embodiment, the method further comprises the steps of: (a) isolating DNA from the maize plant or germplasm; and (b) analyzing the isolated DNA for presence of at least one resistant SNP marker allele on a SNP marker locus wherein the at least one SNP marker locus is located within a maize chromosomal interval comprising and flanked by (i) SEQ IN NO: 15 and SEQ ID NO: 16 on chromosome 8; (ii) SEQ ID NO: 17 and SEQ ID NO: 18 on chromosome 5; or (iii) SEQ ID NO: 19 and SEQ ID NO: 20 on chromosome 3
In one embodiment, the method disclosed herein comprises the steps of: detecting in a maize plant the presence of at least one resistant SNP marker allele on a SNP marker locus wherein the at least one SNP marker locus is located within a maize chromosomal interval comprising and flanked by SEQ IN NO: 15 and SEQ ID NO: 16 on chromosome 8.
In one embodiment, methods for identifying maize plants with enhanced resistance to Exserohilum and/or northern leaf blight by detecting a marker locus in the genome of the maize plant using the sequence of the marker locus, a portion of the sequence of the marker locus, or a complement of the sequence of the marker locus, as marker probe are provided.
In one embodiment, the current invention encompasses a method of selecting a maize plant exhibiting resistance to a norther leaf blight and/or Exserohilum, the method comprising the steps of:
(a) Crossing a donor parent maize plant comprising at least one of the favourable SNP marker alleles as disclosed herein, with a second parent maize plant comprising unfavourable marker allele at the corresponding SNP position in the QTLs to obtain a F1 plant and a segregating progeny F2 plant population by selfing of F1 plant;
(b) Selecting a F2 progeny plant from the segregating progeny F2 plant population with at least one favourable marker allele as claimed herein, in homozygous state in by routine breeding crosses;
In one embodiment, the method as stated above, wherein the second parent plant is a recurrent parent, and the method further comprises the steps:
(a) Backcrossing F1 plant obtained in step (a) of method disclosed above, with the recurrent parent maize plant to get BC1F1 progeny plant population
(b) Selecting a progeny plant from the segregating BC1F1 progeny plant population with at least one favourable marker allele in heterozygous state, and backcrossing selected progeny with favourable alleles with recurrent parent to produce BC2F1.
(c) Repeating step (a)- (b) “n” number of times, wherein “n” is 2 to 6, to obtain BCnF1 progeny plant followed by selfing of BCnF1 plants to get BCnF2 segregating progeny;
(d) Selecting BCnF2 near-isogenic recurrent plant type progeny (segregating for QTL alleles) plants at least with one favourable marker allele as disclosed herein, in homozygous state.
In one embodiment, the current invention encompasses a plant produced by the method as disclosed herein.
In one embodiment, the current invention encompasses a plant exhibiting resistance to NCLB, wherein the plant comprises at least one favourable marker allele as disclosed herein.
In one embodiment, the selected maize plant from the method disclosed herein is homozygous for one SNP marker selected from the group consisting of: SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, and SNP7.
In one embodiment, the selected maize plant from the method disclosed herein is homozygous for two SNP markers selected from the group consisting of: SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, and SNP7.
In one embodiment, the selected maize plant from the method disclosed herein is homozygous for three SNP markers selected from the group consisting of: SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, and SNP7.
In one embodiment, the selected maize plant from the method disclosed herein is homozygous for four SNP markers selected from the group consisting of: SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, and SNP7. In one embodiment, the selected maize plant from the method disclosed herein is homozygous for five SNP markers selected from the group consisting of: SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, and SNP7.
In one embodiment, the screening at step a) and e) is done by standard SNP genotyping methods.
In one embodiment, the current invention encompasses a method of selecting a maize plant or germplasm with enhanced resistance to Exserohilum and/or northern leaf blight.
The method comprises the steps of obtaining DNA from the plants to be tested and the presence of at least one marker allele is detected. The marker allele can include any marker allele that is linked to and associated with: a haplotype on chromosome 8, comprising a “A” at position no 101 in SEQ ID NO: 1, a “G” at position 101 in SEQ ID NO:3, a “T” at 101 in SEQ ID NO:5, and “A” at position 101 in SEQ ID NO: 7; a haplotype on chromosome 5 comprising an “C” at position 101 in SEQ ID NO: 9, an “T” at position 101 in SEQ ID NO: 11; or a haplotype on chromosome 3 comprising “A” substitution at position 101 in SEQ ID NO: 13 . A maize plant or germplasm that has a marker allele linked to and associated with any of these haplotypes or marker alleles can then be selected. Maize plants identified by this method are also encompassed in the current invention. Progeny and seeds derived from the maize plants selected by this method are also encompassed in the current invention.

EXAMPLES
Example 1: NCLB phenotyping and genotyping
To identify QTLs for NCLB tolerance in maize, we evaluated a set of 159 inbreds over three years and in five field testing locations. All genetic material including one of the best NCLB inbred donor (RL-007) were scored on 1-9 disease scale (1 is susceptible and 9 is resistant) visually based on disease symptom appearance. Phenotype distributions suggested additive genetics across locations and years. All inbreds of the field experiments, were subjected to high-density tGBS® genotyping and a set of 84000 genome wide SNP markers set was used for further trait dissection. tGBS® genotyping-by-sequencing was used since conventional genotyping-by-sequencing (cGBS) strategies have high rates of missing data and genotyping errors, particularly at heterozygous sites. tGBS® is a novel method of genome reduction that employs two restriction enzymes to generate overhangs in opposite orientations to which (single-strand) oligos rather than (double-stranded) adaptors are ligated (Ref 6: Ott et al).
Example 2: NCLB trait QTL discovery
General Linear Model (GLM) is a fixed effects model to test for association between segregating markers and phenotypes. The analysis optionally accounts for population structure using covariates that indicate degree of membership in underlying genetic material. MLM is a mixed model which includes both fixed and random effects. Including random effects gives MLM the ability to incorporate information about relationships among individuals. When a genetic marker-based kinship matrix (K) is used jointly with population structure (Q), the “Q+K” approach improves statistical power compared to “Q” only.. FarmCPU (fixed and random model circulating probability unification) method is a random effect model and markers are selected using the maximum likelihood method. FarmCPU uses a bin based approach to avoid selecting markers from the same locations with bin size and also the number of bins are optimized using the maximum likelihood approach. This model assumes that the causal genes are distributed equally across the genome. However, BLINK (Bayesian-information and Linkage-disequilibrium Iteratively Nested Keyway) model eliminates the assumption to improve statistical power by using the linkage disequilibrium (LD) method. Markers are sorted first with most significantly associated maker on the top as reference, and remaining markers are removed if they are in LD with the most associated marker. Among the remaining markers after this step, the most significantly associated marker is selected as the reference. The process is repeated until no markers can be removed from the analysis.
For finding markers associated with resistance to NCLB, multi-year inbreds field phenotypic data and high-density genotypic data was used for advanced Genome wide association studies (GWAS) (Ref 10: Uffelmann et al) analysis with standard parameters for population structure, kinship, multiple corrections. Before the GWAS, the genotypic data was filtered for 0.05 minor allele frequency to avoid spurious associations, and threshold for QTL significance was identified as at alpha 1. A series of GWAS analysis, involving various trait-association single locus and multi-locus models including GLM (General Linear Model), MLM (Mixed linear model), FarmCPU (fixed and random model circulating probability unification), BLINK (Bayesian-information and Linkage-disequilibrium Iteratively Nested Keyway); (Ref 3: Kaler et al; Ref 1 : Alamin et al), were performed to identify all possible QTLs across years and location.
A set of most consistent QTLs with significant threshold on chromosome-8 (chr-8) (up to 18% R2), chr-5 (up to 12% R2), chr-3 (up to 10% R2) were further shortlisted based on consistency over years and locations, and consistent detection across multiple GWAS models. A representative GWAS QTL association Manhattan plot shown in Fig. 1 Manhattan plot illustrates two properties of GWAS results, first the physical location of SNPs with extreme p-values, and secondly the degree to which a SNP association is corroborated by other nearby SNPs in linkage disequilibrium. All QTL associated significant regions or markers with significant p-value are highlighted with arrow mark on different chromosomes (Fig. 1). Negative log 10 p-values are represented on Y-axis, and chromosome numbers from 1 to 10 are represented on X-axis. Negative log10 P-values are plotted against genome position.
Example 3: NCLB QTL haplotype analysis
Most consistent major QTL on chromosome-8 was subjected to haplotype regression association using individual inbred marker alleles for the QTL locus vs trait phenotype across years and locations. Major allele on chromosome-8 found to be highly significant in haplotype association study. Haplotype differentiation analysis of consistent QTLs on chr-8, chr-5 and chr-3 was also conducted using the main tolerant donor RL-007 (score 7.5) vs four highly susceptible inbreds RL-016, (score 1.4) RL-014 (score 1.4), RL-381 (score 1.1), RL-447 (score 2). QTL haplotypes on three chromosomes clearly differentiated all tolerant lines and susceptible inbreds.
Example 4: NCLB major QTL validation using F2 population
An F2 population with contrast QTL haplotypes derived from inbred lines RL-007 (tolerant) x Rl-014 (susceptible) was used for GWAS QTL validation. 376 segregating F2 population along with tolerant and susceptible parental lines, were evaluated in the field for NCLB reaction. The F2 population along with parental lines was scored for the NCLB disease reaction on 1-9 visual scoring similar to the previous genetic field experiments. SNP markers tightly associated with major chr-8 QTL in GWAS study were synthesized for standard SNP genotyping. Four polymorphic SNP markers (SNP-1 to SNP-4) were individually subjected for marker-trait association using haplotype regression analysis with field phenotypic data and SNP marker alleles. All markers, with favourable allele from RL-007 parent, showed significant association with field phenotype with R2 ranging from 28 to 35% in regression analysis. F2 data indicated recessive nature of the QTL reaction. Best marker haplotypes associated with NCLB field phenotype in homozygous classes are represented in Table-2.
Table-2: Chromosome-8 chromosome QTL haplotypes associated with NCLB resistance in F2 population
F2 Field score SNP-1 SNP-2 SNP-3 Disease Class Allele for breeding selection
8 A:A G:G T:T Res Fav
8 A:A G:G T:T Res Fav
8 A:A G:G T:T Res Fav
1 C:C A:A G:G Sus Un fav
1 C:C A:A G:G Sus Un fav
1 C:C A:A G:G Sus Un fav

Example 5: NCLB QTL haplotype analysis for RL-007 derived tolerant lines
To identify the QTL reaction in all inbred lines derived from the donor line RL-007 through field selections, all derived inbred lines either tolerant or susceptible NCLB field phenotype were analysed for the marker haplotypes at major QTL on chr-8. We could find clear presence of tolerant and susceptible haplotypes matching against multi-year field phenotypes.
,CLAIMS:We Claim:

1. A SNP marker for identifying a maize plant or maize germplasm exhibiting resistance to northern leaf blight or Exserohilum, wherein the SNP marker comprises presence of a favourable SNP marker allele at a SNP marker locus and wherein the favourable SNP marker allele is selected from the group consisting of : SNP1 which comprises a C to A nucleotide substitution at position no 101 in SEQ ID NO: 1(RLMZNB8), SNP2 which comprises an A to G substitution at position 101 in SEQ ID NO: 3 (RLMZNB9); SNP3 which comprises a G to T substitution at position 101 in SEQ ID NO: 5 (RLMZNB10); SNP4 which comprises T to A substitution at position 101 in SEQ ID NO: 7 (RLMZNB11) , SNP5 which comprises T to C substitution at position 101 in SEQ ID NO: 9 (RLMZNB1) ; SNP6 which comprises C to T substitution at position 101 in SEQ ID NO: 11 (RLMZNB2) and SNP7 which comprises C to A substitution at position 101 in SEQ ID NO: 13 (RLMZNB12).
2. A method of identifying a maize plant or germplasm that exhibits resistance to northern leaf blight or Exserohilum, the method comprising the steps of:
detecting in a maize plant the presence of at least one favourable SNP marker allele as claimed in claim 1 , wherein the favourable SNP marker allele is associated with resistance to northern leaf blight.
3. The method as claimed in claim 3, wherein the method further comprises the step of
a. obtaining DNA from the maize plant or germplasm; and
b. analysing the DNA from step (a) for presence of any of the SNP markers as claimed in claim 1.
4. The method of identifying a maize plant or germplasm that exhibits resistance to northern leaf blight or Exserohilum, wherein the method comprises the step of detecting in a maize plant the presence of a favourable SNP marker allele selected from the group consisting of SNP1, SNP2, SNP3 and SNP4 as claimed in claim, wherein the favourable SNP marker allele is associated with resistance to northern leaf blight.
5. A method of selecting a maize plant or germplasm that exhibits resistance to northern leaf blight or Exserohilum, the method comprising the step of selecting the plant identified by the method as claimed in claim 3.
6. A method of identifying a maize plant that exhibits resistance to northern leaf blight or Exserohilum, the method comprising the steps of:
(a) detecting in a maize plant at least one QTL or marker locus associated with resistance to northern leaf blight and/or Exserohilum, wherein the at least one QTL comprises:
i. a first QTL (QTL1) comprising at least one SNP marker allele at a locus within a maize chromosomal interval comprising and flanked by SEQ IN NO: 15 and SEQ ID NO: 16 on chromosome 8,
ii. a second QTL (QTL2) comprising at least one marker allele at a locus within a maize chromosomal interval comprising and flanked by SEQ ID NO: 17 and SEQ ID NO: 18 on chromosome 5; or
iii. a third marker locus comprising a marker allele at a locus within a maize chromosomal interval comprising and flanked by SEQ ID NO: 19 and SEQ ID NO: 20 on chromosome 3.
7. A method for identifying a maize plant or germplasm comprising the first QTL, the second QTL or third QTL linked to resistance to northern leaf blight and/or Exserohilum as claimed in claim 6, wherein said method comprises the following steps:
a. providing a sample of genomic DNA from a maize plant or germplasm; and
b. detecting in the sample of genomic DNA the presence of at least one SNP marker linked to:
i. the first QTL wherein said SNP marker is selected from the group consisting of SNP1, SNP2, SNP3, and SNP4;
ii. the second QTL wherein said SNP marker is selected from the group consisting of SNP5 and SNP6; or
iii. the third marker locus wherein said SNP marker is SNP7.
8. A method of identifying a maize plant that exhibits resistance to northern leaf blight or Exserohilum, the method comprising the steps of:
a. detecting in a maize plant the presence of at least one resistant SNP marker allele on a SNP marker locus wherein the at least one SNP marker locus is located within a maize chromosomal interval comprising and flanked by
i. SEQ IN NO: 15 and SEQ ID NO: 16 on chromosome 8;
ii. SEQ ID NO: 17 and SEQ ID NO: 18 on chromosome 5; or
iii. SEQ ID NO: 19 and SEQ ID NO: 20 on chromosome 3
9. The method as claimed in claim 8, wherein the method comprises the steps of:
a. detecting in a maize plant the presence of at least one resistant SNP marker allele on a SNP marker locus wherein the at least one SNP marker locus is located within a maize chromosomal interval comprising and flanked by SEQ IN NO: 15 and SEQ ID NO: 16 on chromosome 8.

10. The method as claimed in claim 8, wherein the method further comprises the steps of:
a. isolating DNA from the maize plant or germplasm; and
b. analyzing the isolated DNA for presence of at least one resistant SNP marker allele on a SNP marker locus wherein the at least one SNP marker locus is located within a maize chromosomal interval comprising and flanked by
i. SEQ IN NO: 15 and SEQ ID NO: 16 on chromosome 8;
ii. SEQ ID NO: 17 and SEQ ID NO: 18 on chromosome 5; or
iii. SEQ ID NO: 19 and SEQ ID NO: 20 on chromosome 3